JPS60155127A - Preparation of biologically active substance - Google Patents

Abstract

PURPOSE:To obtain advantageously a biologically active substance such as remedy for diabetes, mold vaccine, etc., by cultivating a bacterium belonging to the genus Bordetella in a specific madium containing Casamino acid, ascorbic acid, and gluthathione stably and efficiently. CONSTITUTION:A bacterium such as Bordetella pertussis, Bordetella parapertussis, etc.) belonging to the genus Bordetella is cultivated in a medium containing 0.1-20/l Casamino acid, 0.01-1g/l ascorbic acid, and 0.1-5g/l gluthathione, and a biologically active substance is collected from the culture. Well-known Stainer.Scholte medium, SSA medium, etc. are preferable as the medium. Advantageously, 0.001-5g/l cyclodextrin or its derivative besides the components is added to the medium. A mold is stably and efficiently grown, to obtain advantageously an active substance such as insulin secretion enhancing active substance, LPF-HA, etc. of vaccine component of Bordetella pertussis, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Bordetella (B.
ord@tel1m) 174 K microorganisms are cultured using a specific medium, and biological activities a,
! Concerning the method of manufacturing quality.

: Microorganisms belonging to the Bordetella genus include Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.In particular, Bordetella pertussis has a characteristic cough that persists for 100 days. It is an acute infectious disease caused by 68 Illness, the king of coughs! , , is known as f/M. Therefore, rapid and reliable production of such clinical isolates is desirable, and for this purpose, a culture method or culture medium that promotes the growth of clinical isolates is required. . In addition, pertussis vaccine (whole body vaccine or component vaccine) is used for the prevention of cough, but the effectiveness of the vaccine is low.
to write. For pertussis 1) A proper culture method or culture medium that promotes growth is desirable.

In other words, 100 days - Expansion of bacteria culture (integrated with culture medium) into a drug for treating and preventing urine disease - Potassium 1.
After waiting 15 minutes, I took insulin for 15 minutes.
n.

(hereinafter abbreviated as IAP) and 1eukocytosi, which is attracting attention as a vaccine component for C day@-.
s promoting factor-hsmagg
lutinin (hereinafter abbreviated as LPF-HA) and fi
lamentous-hemagglutinin (
Medically effective biologically active substances such as F-)) (hereinafter abbreviated as IA) can be obtained, but in order to efficiently produce such biologically active substances, an efficient culture method for Bordetella pertussis is required. be.

As a medium for the growth of brains belonging to the genus Bordetella, especially Pertussis I, Cohen et al.
rican Journal of Public)
leath36 371-376 (1946) N, Sutherland et al.
Hology and Bacteriology 82
431-43 B (1961)) is known, which contains activated carbon, starch, or ion exchange resin. However, activated carbon and ion exchange resins form a heterogeneous system, making them unsuitable for tracking changes in bacterial growth over time.Also, because they have nonspecific adsorption properties, they do not absorb factors that inhibit bacterial growth, such as fatty acids. The removal effect cannot necessarily be said to be selective. The effect of starch on bacterial growth was not sufficient, and there was a desire to develop a new culture medium that would compensate for the above deficiencies. In addition, when clinically separating B. pertussis as a single colony, it is possible to directly contact the daily spray solution of pertussis patients with an Okiya agar medium containing blood, such as Poldey-Zhang medium (hereinafter abbreviated as SS medium). However, these media have the drawbacks of poor storage stability and poor reproducibility of colony forming ability since they contain fresh blood as an essential component. Therefore, from this point of view, it has been desired to develop a clinical separation medium that has a certain chemical composition and has a long shelf life.

In recent years, Steady (5tainer) and Shiel 7L
Therefore, a synthetic medium for mass culture of pertussis was developed (Journal of General Microbiology (J, gen,
63, pp. 211-220.

(1971). This Steady-Sholte medium (hereinafter S
S medium (abbreviated as S medium) does not contain additives such as blood and polypeptone derived from natural products that may cause variations in the amount of bacteria, so the culture medium composition of the bacteria can be strictly controlled, making it possible to control the bacterial properties. without causing any changes (can be cultured,
During the separation and purification of biologically active substances such as IAP or LPF-HA mentioned above, pertussis vaccines and biological studies from B. Although it is widely used to produce clinically active substances on an industrial scale, the production of LPF-)LA is insufficient under stirring or static liquid culture conditions (and the inoculation size is 10' cellg). /1 or less, it has the disadvantage that stable growth characteristics cannot be obtained.Also, an agar medium (hereinafter abbreviated as SSA medium) obtained by adding 1 to 2% agar to SS medium and solidifying it has the disadvantage that stable growth characteristics cannot be obtained. In this case, seeding of less than 10' calls (no cominie formation in geatons is observed) has a large defect.

The purpose of the present invention is to produce a biologically active product by culturing a microorganism belonging to the genus Bordetella using a medium that can stably and efficiently culture the microorganism belonging to the genus Bordetella. Our goal is to provide the following.

The purpose of the present invention is to suppress the microorganisms belonging to the genus Bordetella, from 0.1 to 2011/], and to prepare 7scorbic acid from 0.01 to 1 Jl/7! and glutathione in a medium containing 0.1 to 5 N/l, and the biologically active substance is collected from the culture (culture medium and box). Achieved through manufacturing methods.

In the present invention, microorganisms belonging to the genus Bordetella refer to Bordetella pertussis, Bordetella pertussis, and Bordetella bronchiseptica. In the present invention, Bordetella pertussis is preferably used, and Pertussis I phase is particularly preferred. Regarding the dental properties and growth conditions of microorganisms belonging to the genus Bordetella, please refer to B@rg
)”Manual of Det@rminative
Bacteriology + 8th edition, 1974,
The Williams & WillkinsC
o0 Publishing Ya J, Exp Med, Volume 129, No. 523
- 550 pages, 1969 or #1 Gypology Practice Summary, 3rd edition, 80 pages and below, 1971, published by Maruzen, etc., and is already publicly known.

The casamino acids used as a component of the culture medium of the present invention are acid hydrolysates of casein and can be prepared manually as a dry powder. Furthermore, ascorbic acid is known as vitamin C, and glutathione is a type of peptide that is widely distributed in yeast, animal liver, muscle, and the like.

Glutathione may be in an oxidized form, a reduced form, or a mixture thereof. The culture medium of the present invention preferably contains casamino acids of 0.1 to 20 y/Im (), 5
~10 Jl/l, ascorbic acid 0.01-1
jl/11, preferably 0.1~(), 41/II
It is necessary that the amount of glutathione is 0.1 to 511/1, preferably 0.1 to 111/l. If it is outside the above range, the object of the present invention will not be fully achieved.

When the culture medium of the present invention contains, in addition to the above-mentioned components, a cyclotext rinse containing 0.001 to 5&/II, preferably 0.05 to 5&/II of a derivative thereof, the objects of the present invention can be more advantageously achieved. Ru.

Cyclodextrin or a derivative thereof may be added to the medium containing the above-mentioned components as it is and mixed therewith, but it is also possible to form a clathrate compound with glutathione in advance and then add this clathrate compound to the medium. Cyclotextrin in the present invention means α, β, r cyclodextrin, and cyclodextrin derivatives include
Aminated derivatives such as dextrin and 7-minodeoxycyclotextrin, 7-cetylcyclo-a-dextrin and 2-l
-4 esterification of cyclodextrin, methyl cyclotextrin, ethyl cyclodextrin,
Propylcycq-dextrin, carboxymethylcycq-a
Etherified fat derivatives such as dextrins (hereinafter referred to as etherified cyclotextrins) are preferred in the present invention, and among them, hexakis(2,6-o-dimethyl)α-cyclotextrins are preferred. Particularly preferred are methine kistrins, such as (Med-CD) and heptakis(2,6-o-dimethyl)β-cyclodextrin (Meβ-CD).

In the present invention, the medium means a conventionally known liquid medium such as Rayon or peptone water, or a conventionally known solid medium prepared by adding agar, gelatin, egg white, 1 serum, etc. to a liquid medium. Preferred are 88 medium (or 88B medium) and SSA medium, which is solidified by adding about 1 to 2% (w/v) agar thereto. For example, 5SJa
The base is usually around 11, monosodium glutamate.

! - Proline, sodium chloride, potassium phosphorus #2 hydrogen, potassium chloride, magnezium chloride λ.

Calcium chloride and trisphthalaminomethylaminomethane were added to 1θ and 7. 0,24. 2,5°0.5
．． 0.2. 0.1. 0.02. 1.525 I
Adjust the aqueous solution containing I to pH 7.6 with concentrated hydrochloric acid! After 4 adjustments,
Sterilize in an autoclave at 124°C for 15 minutes [obtained]
In the basal medium, l-instin, ferrous acid, ascorbic acid, niacin2. Glutamic acid 1. 4 and 1 per J, respectively. ．． 2. (1,4, I O & 1,000 qb (v/v) per 4 dl of the base medium.
), , is added at a rate of ℃, which is obtained. In the present invention,
To such a medium are added necessary amounts of casamino acids, 7-scorbin and glutathione, and in some cases, a cyclotext rinse is added to the required amount of the derivatives. The violet is cooled.

The culture medium of the present invention has a stable growth rate of 54 J, and can be preferably used for clinical isolation, growth and detection of pertussis. It can also be used as a diabetes treatment mulberry
IAP, which is expected to be used as a medicinal agent in the future, and LPF-1-1A, which is expected to be a component of the white sun cough vaccine.
It is also an extremely advantageous medium for the production of active substances such as PF-)IA and integrated vaccines.

The culture method and conditions for microorganisms belonging to the genus Bordetella used in such a medium are not particularly limited (although conventionally known methods and conditions can be adopted, but shaking and
Cultivation is preferable, and a suitable culture temperature is 30 to 38° C. and a culture time of 10 to 100 hours.

Method 1 means for collecting produced biologically active substances from a culture (culture medium and bacterial cells) is not particularly limited, and known method 2 means can be used. For example, LPF
- To obtain HA, Bordetella batasis Higashihama strain was cultured in the medium of the present invention at 35'G for 48 hours, and the centrifuged supernatant (pH 8.3) of the resulting culture was , pH
Pass through a hydroxyapatite column equilibrated with 8.0 o, 01M IJ acid buffer. and,
After adjusting the resulting permeate to pH 6.0, this time it was adsorbed onto a hydroxyl 7-batite column equilibrated with O, LIIMIJ acid buffer at pH 6.0, and this was adsorbed onto a hydroxyl 7-batite column containing U', 5M sodium chloride. A protein fraction is obtained by elution with 0.1M phosphate buffer (pH 7.0). This protein fraction was adsorbed on affinity' chromatography using haptoglobin-Sepharose 4B as a support.
L
P#'-)IA can be obtained.

In addition, the adsorbed material on a pH 8.0 hydroxy 7-batite column was mixed with 0.5 M sodium chloride. I
A protein fraction is obtained by elution with M phosphoric acid (pH 7,0). This protein fraction is allowed to pass through affinity-take a mass spectrometry using haptoglobin Sephas 4B as a support, and F-klA can be obtained from the passed-through solution.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Normal SSA medium (ascorbic acid 0.021-/
The concentration of glutathione in the solution was increased by 1.5 times, that is, by 0.1 times.
151/l K was changed, and the concentration of Casamino Kai was changed to 0.
．． 0.5. 1.0. 2,5. 5. (1°10.
011/l, and the concentration of Meβ-CD is 13.0/l. 0,05. 0,1 (1,t), 25. 0
．． Improvement 8 with 14 adjustments to make it 50゜1.01/II
Approximately 100 pieces of the 100 Days @ I Phase Kaihama strain were spread on 8A medium and cultured at 35°C for 3 days. The number of colonies formed was compared with the number of colonies formed in the flG medium under the same conditions. The results are shown in Table 1. M
eβ-CI) from 0.50 to 1. It can be seen that almost the same number of colonies were formed when BG medium dJ was added to the BG medium. Casamino acids 085-5
．． (The presence of #/l made the appearance of Koe + Knee more obvious.

Table 1 BG medium for comparison = 104±16 colony count/plate *: Colonies are small.

Example 2 &t! of normal SSB medium! Add 1o1 of casamino acids to the medium.
In addition, 1.5 times glutathione and 20@I/C of glutathione were added at a ratio of 1:1 and 7:1/l (V/V). The thus obtained improved ssB medium (Kaza 4/1i#!'):
10! ' / j e 7 s = ruby 7m = 0
．． 411/l (contains glutathione 0.15.9/l), and Meβ-CD at 0.000/l. 0,05. 0.5.
2.0°s, oM/l, and after dispensing each 200- to a Sakaro flask, inoculate with Higashihama strain G Hiccough I phase at 1.5 x 10°11111/m. , and cultured at 35°C. The results are shown in Figures 1 and 2.

FIG. 1 shows the relationship between 1llll chastity (IOU/1lj) and item number i1, and FIG. 2 shows the time course of LPF-)IA activity. It can be seen that when the medium of the present invention is used, the proliferation of pertussis and LPF-)IA production are accelerated. and,
It can also be seen that this effect is promoted by the departure of Meβ-CD.

Note that #i concentration and 1. PF-)IA activity was determined as follows. -The concentration is the turbidity of the medium-jII solution (0
Dsse) was measured. 10υ and Interna
It is an abbreviation for tional 0pacity unit and corresponds to 110U=10°/1. Lpy-KA activity was determined by collecting LPF'-)IA produced by the following method and adding the activity by 11. Centrifuged supernatant of culture solution (p)
18.6) was passed through a hydroxyapatite column equilibrated with a 1,01M phosphate buffer (p) of 18.0, and the resulting permeate was adjusted to pH 6, OK. (Equilibrated with 11M phosphate buffer, adsorbed on a "Yin Hydoku" xiapatite column, eluted with 1M solution of 0.1M phosphoric acid containing 5M sodium chloride (p) 17.0). A protein fraction was obtained. This protein fraction was adsorbed with haptoglobin-Sepha IJ-sulfate on a support and 1°C in 1E chromatography, and 0.5M NaCA! and 3M potassium thiocyanide were added. LPF-)IA was obtained by desorption with 0.1 M Tris buffer containing LPF-)1A.
T8 sex was determined using the #prime antibody method (ELISA method, No. 28) of Sahara et al.
Micetoxin Symposium (July 23-24, 1981,
(See Iwate Prefecture to Hathira) Collection of Lecture Abstracts, Nos. 141-144)
The activity unit (u) of LPF-)1A is
OD40 (Imμ is 11 per unit volume tk (WLt)
．． The dilution factor of each sample was calculated to give a value of 1.

Example 3 7Scorbic acid, glutathione in the same manner as in Example 2 (but without adding M.β-CI).

Prepare a medium containing casamino acids at once and carry out 11
Bordetella pertussis was cultured using IJkIm in the same manner as in 2, and the LPF-)IA activity was measured 48 hours later.

The results were as shown in Table 2.3.4 - Table 2 Reduced layer glutathione: u, ls &/1 casamino acid
", 10Jl/1 Table 3 ■ 7♂ Corbin Ill: 0.41/1 Casamino acids: 101/1 Table 94 Ascorbic acid: 0,41/1 Glutathione v: u, 1sl/1 Example 4 (1) Culture Normal basic SSB medium 1 tcxoll/1 of casamino acids and 21/l of Meβ-CD were added, and to this was added 1.5 times the standard composition of glutathione and 20/l of ascorbic acid.
A replacement fluid that had been doubled in volume was added at a ratio of 1.0% (V/V). The improved SSB medium thus obtained!
5. Inoculate pertussis ■phase open to 1.0 × 10° cells/d. Culture was carried out using an Ol layer plater.

After 48 hours, vMd degree H is approximately 10'J161/TLl,L
P F-)i A is 12500/W directly by ELISAI
It became Lt. The supernatant (3, 61) obtained by centrifuging the culture solution for 6000 rvaXBU was used in the following L P
It was used for F-HA purification.

(2) Separation and Purification of LPF-HA from Culture Strain Culture supernatant 3.61 (pH 8.3) was purified at around 4°C (1, (11
The mixture was passed through Heid a xylapatite (Cicolumn 001113, manufactured by BDH Chemicals) equilibrated with molar phosphate buffer (p)i 8 ) at a flow rate of 200 M1/hour. υ, (IIMIJ) Wash the column with cough buffer (pH 8) 0.41, and adjust the pH of the resulting effluent 4601 with hydrochloric acid.
Adjusted to 6.0. This solution was mixed with o, o IM phosphorus #
i, Inject IUc 1 m into a hydroxylapatite column (140#+7) equilibrated with a buffer solution (pH 6,0).
(Run for 7 hours. 0. OIM phosphate buffer g, (pH 6
, 0) 0.51, then washed with 250 ml of 0.1 molar phosphate buffer (pH 7,0), and further washed with 0.51 ml of 0.1 molar phosphate buffer (pH 7,0).
0.1 molar phosphate buffer containing 5 molar saline (p)17.
0) and fractionated into 2 fractions. Collect the desired protein fraction (1
00111) Butoglobin-Sephas 4, which was blunted with 0.1 molar phosphate buffer containing 0.5 molar salt
Passed through column B (30aJ). The adsorbed material is 3
One fraction was eluted with a 0.1 molar phosphate buffer containing 0.5 molar potassium perthiocyanate and 0.5 molar sodium chloride. The obtained protein fractions were collected and dialyzed against a 0.1 molar phosphoric acid solution (pH 7.0) diluted with 0.5 molar sodium chloride to obtain LPF-j (A at 20%).
I got 7 da.

This product contains polyacrylamide gel (polyacrylamide concentration 7.5%, INKOI (-Rice Vinegar I)
(pH 4,37) Disc electrophoresis showed a single band, and the migration position matched that of existing PF-HA. Also, the kLlsA value of this product is 111. It was IU/μJ.

[Brief explanation of the drawing]

Figure 1 shows the culture medium of the present invention? When used Q) This is a diagram showing the relationship between phlegm degree and culture time, and Figure 1,1142 is the same <LP
F-) It is a diagram showing the relationship between fA activity and culture time. Figure 1 0 12 24 J6 4δ Arrival time ()II-) Continued from page 1 0 Inventor Akira Ono 4-chome Asahigaoka, Hino City Inventor Hisao Yamaguchi 4-chome Asahigaoka, Hino City Laboratory No. 3-2 Teijin Ltd. Biomedical Research No. 3-2 Teijin Ltd. Biomedical Research

Claims (1)

[Claims] 1. Zero Casamino, a microorganism belonging to the genus Bordetella.
．． 1-2011/1,7 Scorbic acid 0.01. ~1
1//1. A method for producing a biologically active substance, which comprises culturing in a medium containing 0.1 to 5ℓ of glutathione and collecting the biologically active substance from the culture. 2. In addition to casamino acids, 7-scorvin acupuncture, and glutathione, cyclodextrin or snow conductor 0.
The method for producing a biologically active substance according to claim 1 of the patent application, characterized in that it contains 001 to 59/l.